Fig 2: CYLD depletion rescues defective M1-ubiquitylation in Complex I and consequently protects A20-deficient cells from TNF-induced apoptosis.a A20+/+ and A20-/- MEFs were transfected with siRNA targeting CYLD or non-specific siRNA (NS) and stimulated with 1 µg/ml FLAG-hTNF for the indicated duration. TNFR1 Complex I was subsequently FLAG-immunoprecipitated. Protein levels were determined by immunoblotting. b–d A20-/- MEFs were transfected with siRNA targeting CYLD or nonspecific siRNA (NS). Cells were pretreated with the indicated compounds for 30 min before stimulation with 10 ng/ml mTNF. Cell death markers were monitored by immunoblotting (b) and cell death was measured in function of time by SytoxGreen (SG) positivity (c, d). e A20-/- HaCaTs were transfected with siRNA targeting CYLD or nonspecific siRNA (NS). Cells were pretreated with CHX for 30 min before stimulation with 10 ng/ml hTNF. Cell death was measured in function of time by SytoxGreen (SG) positivity. f, g A20+/+ Cyld+/+ (n = 3), A20?IEC Cyld+/+ (n = 3), and A20?IEC Cyld ?IEC (n = 3) were injected i.p. with 5 µg/20 g mTNF. Caspase activity was quantified from small intestinal tissue homogenates 2 h post injection using a fluorescent caspase-activity probe (DEVD-AMC) (f) or via immunoblotting (g). h, i A20+/+ Cyld+/+ (n = 5), A20?IEC Cyld+/+ (n = 4), and A20?IEC Cyld ?IEC (n = 5) were injected i.p. with 5 µg/20 g mTNF. Cumulative survival rates (h) and body temperature (i) were determined in function of time. Temperatures are presented as mean ± SEM. Statistical significance for body temperatures of the mice was determined using two-way ANOVA followed by a Tukey post hoc test. Survival curves were compared using log-rank Mantel–Cox test. Cell death experiments are presented as mean ± SEM of three independent experiments. Statistical significance for the cell death assays was determined using two-way ANOVA followed by a Tukey post hoc test. Significance between samples is indicated in the figure as follows: *p < 0.05; **p < 0.01; ***p < 0.001; ns non significant.

Fig 3: Figure 5. MALT1 auto-proteolysis in activated B cells.A) ABC-DLBCL cell lines HBL-1 and OCI-Ly3 were treated with 50 µM z-VRPR-fmk (36 hrs hrs) and lysates were analysed for the presence of MALT1 and BCL10 cleavage fragments with a-MALT1, a-Cleaved BCL10 and a-Tubulin (loading control). B-C) The GCB-DLBCL cell lines BJAB and Raji were left untreated or stimulated with PMA/ionomycin (30 min min) with or without pre-treatment with 50 µM z-VRPR-fmk (30 min min). Lysates were analysed for the presence of MALT1 and BCL10 cleavage fragments, for p-ERK (activation control) and tubulin (loading control). D) Immunoblot of lysates of SSK41 cells and SSK41 cells with ectopic expression of the API2-MALT1 fusion variants A7M3 and A7M8, or the L232LI mutant of Card11 (C11m) respectively, with antibodies against the MALT1 C-terminus, the p76 neo-epitope, the CYLD C-terminus, the NIK C-terminus and Flag (ectopic A7M3, A7M8 and C11m). Numbers below blots depict band intensities of MALT1, p76 and the CYLD p70 fragment relative to lane 1. * = non-specific band. LC: loading control, a non-specific band obtained with the p76 neo-epitope antibody was used.

Fig 4: CYLD is phosphorylated in an IKKe/TBK1 dependent manner, which can be detected by immunoprecipitation of CYLD using the phospho(Ser418)-CYLD-specific antibody. (A) Wild-type (wt) or IKKe/TBK1 double knockout (ko) Jurkat T cell clones were stimulated with 200 ng/ml PMA and 1 µM Ionomycin (PMA/I) for 30 min. (B) Wt Jurkat T cells were pre-incubated for 30 min in the presence of 2 or 10 µM IKKe/TBK1 inhibitor MRT67307 and then stimulated with 200 ng/ml PMA and 1µM Ionomycin for 30 min. (A,B) Cell extracts were subjected to immunoprecipitation using the phospho(Ser418)-CYLD-specific antibody. Beads without antibody were used as negative control (nc). Specific proteins were determined by immunoblotting with the indicated antibodies.

Fig 5: The phospho(Ser418)-CYLD specific antibody still detects a protein of the same size as CYLD in CYLD-deficient cells. (A) Wild-type (wt) or three different CYLD knockout (ko) Jurkat T cell clones were stimulated with 200 ng/ml PMA and 1 µM Ionomycin for the indicated times. (B) Jurkat cells or (C) primary murine CD4+ T cells were stimulated with 200 ng/ml PMA and 1 µM Ionomycin (PMA/I) for the indicated times. Cell extracts were subjected to immunoprecipitation using the phospho(Ser418)-CYLD-specific antibody. Beads without antibody were used as negative control (nc). (A–C) Specific proteins were determined by immunoblotting with the indicated antibodies.